Method of decontamination of whole structures and articles contaminated by pathogenic spores

ABSTRACT

The present invention is a method of decontaminating a structure contaminated by pathogenic microorganisms such as  Bacillus anthracis  and its spores,  B. subtilis  var  niger  and its spores, and  B. stearothermophilus  and its spores, comprising the steps of sealing a contaminated structure sufficiently to enable retention of a gas, introducing methyl bromide gas into sealed contaminated structure to a concentration of methyl bromide in an amount sufficient to deactivate said pathogenic microorganisms and disable germination of pathogenic bacteria spores, and maintaining said sealed contaminated structure with said concentration of methyl bromide at a sufficient temperature for a sufficient period of time, and deactivating said pathogenic microorganisms and disabling germination of said pathogenic bacteria spores associated with said contaminated structure. The method is performed approximately in the range of 20° C. to 40° C., and the concentration of methyl bromide is about 200 mg/l to 303 mg/l during the decontamination. High humidity levels are preferred.

This patent application claims priority to and the benefit of U.S.patent application Ser. No. 60/346,282 filed on Jan. 9, 2002.

BACKGROUND OF THE INVENTION

1. Field of Invention

The instant invention relates to a novel method and means for theefficient, safe, and economic decontamination of entire physicalstructures, such as buildings, and/or articles contaminated bymicroorganisms inclusive of pathogenic spores.

2. Prior Art

The prior art of eradication of entire buildings of insects such astermites, cockroaches, wood-boring beetles, rats, mice, bats and othersuch arthropod and vertebrate animals has, over the last approximatelysixty years, consisted of covering and sealing the structure to betreated with a vinyl tarpaulin and, thereafter, introducing the selectedfumigant into the structure covered by or confined within the tarpaulin.Among the various fumigants used in the prior art for this purpose hasbeen methyl bromide, a USEPA registered pesticide. However, due to theozone-depleting properties of methyl bromide as have been recognized bythe Montreal Protocol of 1992, methyl bromide was officially added tothe list of ozone-depleting chemicals and, pursuant thereto, itsproduction was frozen at 1991 levels thereof. Further, any chemical withan ozone depletion potential (ODP) of greater than 0.02 was, under theProtocol, banned at the end of 2000 and, in the case of methyl bromide,its use in the United States has been banned by the EnvironmentalProtection Agency (“EPA”) effective in 2005. Moreover, neither methylbromide nor other known structural fumigants, have ever been employedfor the purpose of decontamination of a structure which are contaminatedby microorganisms that are pathogenic to humans including bacteria(prokaryota).

Methyl bromide has been used as a fumigant since the 1930s to controlpests in soil, stored commodities, structures and shipments that mustmeet agricultural quarantine regulations. The major use in agricultureis soil fumigation. It is an extremely effective herbicide, nematicide,insecticide, and fungicide. It is important for the large-scalecommercial production of strawberries, tomatoes, peppers, melons,grapes, cucumbers, eggplants, ornamentals, and tobacco. In addition,U.S. regulations require that a wide array of imported food and non-foodcommodities be fumigated with methyl bromide as a condition of entryinto the country. Methyl bromide has never been employed as a means fordecontamination of structures of human pathogens such as viruses andhazardous bacteria. Rather, if the contamination of a structure wasknown to be limited to a particular surface or article, one could employbleach (sodium hypochlorite), a foam, such as Sandia foam, or a toxicgas, such as chlorine dioxide, which are commonly unstable and/orexplosive.

Methyl bromide (CH₃Br, also abbreviated as MeBr), a member of thechemical family of alkyl halides, is a colorless and non-flammable gas,which has no odor at toxic levels. It is stable under normal conditionsof handling and use. Methyl bromide gas is commercially available at apurity greater than 99% from Great Lakes Chemical Corporation (WestLafayette, Ind.) under the trade name Meth-O-Gas® 100 and Meth-O-Gas®Q.Most fumigation treatments are recommended on the basis of dosage fora certain volume, expressed in pounds per 1000 cubic feet (lb/1000 ft³)in the industry, and in milligram per liter (mg/l) using the metricsystem. The common dosages used for treating commodities range from 1lb/1000 ft³ for dried fruits such as dried apples and prunes, to 8lb/1000 ft³ for cotton seeds, equivalent of from 16 to 128 mg/l. Forstructural fumigations, such as in termite infested houses andbuildings, the common dosage is from 1 to 3 lb/1000 ft³, equivalent offrom 16 to 48 mg/l. An important factor used in the industry is theamount of gas acting on the pests over a certain period of time,expressed as the product of concentration and time (CT product,mg-hr/l). The use of methyl bromide has never been considered at theconcentration, temperature, and time combinations suggested herein.

Given the new reality of bio-terrorism and its potential in the wake ofthe events of September, 2001, much concern and attention have beendirected to the decontamination of entire buildings that have been thesubject of a biological attack such as occurred in Florida, Washington,D.C., and various U.S. Post Offices in New York, New Jersey, Connecticutand elsewhere. Government, inclusive of the EPA and CDC, as well asresearch community and industry have lacked experiences other than thatof decontaminating buildings subjected to a purely chemicalcontamination, such as by asbestos or PCBs and therein has had noexperience with the many problems related to the decontamination ofentire buildings, parts of which have been infected (or potentiallyinfected) by a bio-weapon consisting of a microorganism such as a virus,bacteria or spores thereof. Historically, structural decontaminations ofbiological weapons occurred at an Army facility at Ford Detrick in 1970and 1971 for a building previously used for producing Bacillusanthracis, commonly known as anthrax. The building was decontaminatedtwice using formaldehyde gas, and further hydrochloric acid was forcedthrough pipes and valves that carried a bacteria mixture. Anthrax sporesand other bacteria were not found after the decontamination. However,the damage by the caustic decontamination has threatened the structuralintegrity of the building. In light thereof, the EPA and CDC, afterconsultation with scientists, public health specialists, industryexperts and even historians on the subject of bio-weapons, settled onthe use of chlorine dioxide gas on a massive scale, never beforeattempted, as a means of decontaminating buildings exposed to abio-weapon and, particularly, spores of anthrax. As such, operating withlittle historical or scientific precedent, EPA/CDC have attempted toemploy, in substantial quantity, chlorine dioxide gas which, in thepast, had been employed only in context of purification of drinkingwater. In these efforts, the EPA determined that use of chlorine dioxidegas on a large scale entails several hazards and problems, this apartfrom an underlying question with regard to the concentration,temperature, humidity and time of exposure necessary to kill anthraxspores in sufficient quantity. For example, the humidity within abuilding to be so decontaminated must be first be elevated from anambient level of about thirty percent to that of seventy percent, whichis considered the optimum level for chlorine gas to kill spores ofanthrax. Furthermore, because of the extreme oxidative properties ofchlorine dioxide, substantially all equipment and furniture, within abuilding to be decontaminated must be removed to preclude corrosion ordegradation of the surfaces thereof by the oxidative effect of thechlorine dioxide. Further, a special chemical, such as sodium bisulfate,must be employed to neutralize and vent the chlorine dioxide from thebuilding itself. Then, all removed equipment and furniture must bebrought back into the building after being fumigated in chambers withflammable ethylene oxide gas. Thereafter, test strips containing abacterial endospore (Bacillus subtilis var. niger) more chemicallyresistant than anthrax spores are used to verify the effectiveness ofthe chlorine dioxide decontamination.

In view of the above, an urgent need has arisen for a method ofdecontamination of whole buildings (including the inside, outside, ductwork and piping therein,) that will not only obviate the need to removesensitive articles such as photographs, documents, and computers, andheavy items such as furniture, but which will also obviate the need topump steam into the structure to elevate the humidity thereof and thatdoes not require a special chemical to neutralize the active agentbeyond these issues is a need for a method that can provide a higherlevel of confidence that the targeted pathogenic organism has, in fact,been killed, and therefore is unable to germinate.

In spore-forming bacteria, the spore is protected from environmentalextremes of drought and temperature by a coat made of numerouscross-linked proteins (Driks, A. 1999. Bacillus subtillis spore coat.Microbiol Molec. Biol. Rev. 63:1-20). It has been determined that sporecoats or shells exhibit a considerable degree of uniformity in theirchemical linkages, such that the shell of one spore is substantiallysimilar in chemical structure to the shell of other spores and, inparticular, spores of Bacillus which is the genus of a species ofbacteria, namely, B. subtilis var niger, employed as the test bacteriaupon test strips, that has become a universally accepted standard fordetermination of whether a bacterial spore has been killed by a treatingagent or method. These spores and bacteria are also used to test theefficacy of treatments for killing B. anthracis.

The instant invention therefore addresses the need for a more reliable,comprehensive, convenient, and cost-effective method of decontaminationof entire structures and their contents that have been contaminated, orpotentially contaminated with pathogenic microbes including, withoutlimitation, anthrax and spores thereof.

SUMMARY OF THE INVENTION

The present invention comprises methods of using methyl bromide as ananti-microbial agent in the decontamination of whole structures orarticles that have been contaminated, or may be contaminated, withmicroorganisms such as bacterial spores. This invention is based on anew use of methyl bromide, a non-flammable, non-corrosive, and E.P.A.registered pesticide. The instant method entails the gas-tight sealingof the entire building to be decontaminated under tarpaulins or otherequivalent means. After the building is sealed, a sufficient amount ofmethyl bromide gas is introduced within the seal so that methyl bromidediffuses into all parts of the building, its surfaces and articlestherein. The sealed structure is maintained with the concentration ofmethyl bromide at a sufficient temperature for a sufficient period oftime. This process renders pathogenic bacterial spores in or associatedwith the structure or its contents nonviable so that germination willnot occur when the spore is exposed to a favorable environment forgermination. The method further comprises a step of unsealing andaerating decontaminated structure to aerate the methyl bromide gas,without requirement for any special purpose cleaning or deactivationagent, either in gaseous form mixed with air, or as a scrubbingprocedure following the fumigation.

The application of methyl bromide using the method of the presentinvention, at temperature greater than 20° C., preferably greater than25° C., and with a concentration of methyl bromide about 200 mg/l andabove, and an exposure time greater than 30 hours, prevents germinationof B. subtilis var. niger and B. stearothermophilus spores. Inadditional field testing, the application of methyl bromide using themethod of the present invention, at temperature between 28° C. and 38°C., and with a concentration of methyl bromide about 250 to 350 mg/l,and an exposure time of approximately 48 hours, also preventsgermination of these spores. The method of the present inventionprovides a marked improvement in coverage, level of confidence, andcost-effectiveness of result over existing methods of structuralmicrobial decontamination.

It is accordingly an object of the invention to provide an improvedmethod of whole structure decontamination for pathogenic microbes.

It is another object to provide a method of the above type, which doesnot require the removal of all contents of a structure to bedecontaminated or the elevation of the humidity, therein.

It is a further object of the invention to provide a method ofstructural decontamination for pathogenic spores without subjecting theinterior of a building as so treated to oxidative agents or requiringthe use of special cleaning agents to effect removal of the treatingagent at the end of the process.

It is a still further object to provide a method of the above type tomore cost-effectively, safely and reliably decontaminate a structure ofmicrobial pathogens including, without limitation, Bacillus anthracisspores.

It is another object to provide a method of the above type comprising ause of methyl bromide as a decontaminating agent for bacteria and theirspores.

It is a still further object of the invention to provide a method of theabove type in which the effectiveness thereof may be authorativelyascertained through the use of test strips containing bacteria of a typemore resistant to heat and toxic chemicals than B. anthracis and whichare universally accepted as a measure of the effectiveness of microbialdecontamination.

It is a yet further object to provide a method of the above typeemploying long established procedures for fumigation of structures forthe control of arthropod and vertebrate pest animals, however, onlysubstituting the use of methyl bromide in its inventive parameters ofuse, in lieu of existing fumigants.

It is yet another object of the invention to use a chemical fordecontaminating B. anthracis and its spores which is non-flammable andstable.

It is still another object of the invention to provide a method of theabove type comprising a use of methyl bromide as a decontaminating agentfor bacteria and their spores which generally does not harm electronicequipment or sensitive materials which might be damaged by a strongoxidizing agent

It is yet another object of the invention to provide a method of theabove type that allows for an unlimited volume for treating areas.

The above and yet other objects and advantages of the present inventionwill become apparent from the hereinafter set forth Brief Description ofthe Drawings and Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart detailing the methyl bromide concentration duringtrailer fumigation and aeration of Example 4.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to a method ofdecontaminating a structure contaminated by pathogenic microorganisms.The method comprises the steps of sealing a contaminated structuresufficiently to enable retention of a gas; introducing a methyl bromidegas into the sealed contaminated structure at a concentration of methylbromide in an amount sufficient to deactivate the pathogenicmicroorganisms; and maintaining the sealed contaminated structure withthe concentration of methyl bromide at a sufficient temperature for asufficient period of time, resulting in deactivation of the pathogenicmicroorganisms and prevention of germination of the pathogenic bacteriaspores in or associated with the contaminated structure.

For the purpose of the present invention, the term “deactivation” meanskilling pathogenic bacteria and bacterial spores so that germinationwill not occur, in another word, disabled, when the spores are exposedto a favorable environment for germination. The term “structure”includes, but not limited to, a room, a residential or a commercialbuilding, a mobile home, a vehicle, a train, a boat, or an airplane.

Preferably, the methyl bromide gas used in the above-described processis a pure gas. A suitable example is a commercial product under thetrade name Meth-O-Gas® 100 obtained from Great Lakes ChemicalCorporation (West Lafayette, Ind.). The purity of Meth-O-Gas® 100 isgreater than 99%, with trace amount of dimethyl ether and methylchloride.

Furthermore, methyl bromide, as the active agent for deactivatingpathogenic microorganism, can also be used together with otheradditives. Suitable additives include inert carrier gases which do notinterference methyl bromide's function, other gaseous chemicals whichmay further enhance the effect of methyl bromide against themicroorganisms, and warning agents. Suitable examples of additives arecarbon dioxide and chloropicrin, and both are compatible with methylbromide. Since methyl bromide is an odorless gas at a toxicconcentration, it can be desirable in certain circumstances to introducea small amount of warning agent into the methyl bromide gas filledstructure to alert the risk in case of leaking, or before the structureis ready for reentry after decontamination. Chloropicrin, availablecommercially, has been used as a warning agent for fumigation because ofits pungent odor and being an irritating lachrymator. Additionally, amethyl bromide gas containing a small amount of chloropicrin is alsocommercially available. One example is the methyl bromide gas containing2% or 5% of chloropicrin produced by Great Lakes Chemical Corp. underthe trade name of Brom-O-Gas®.

Efficacy of the process of decontaminating a structure contaminated bypathogenic microorganisms can be tested by using spore strips. Prior tothe start of the fumigation bacterial spore strips containing 10⁶ ((1million spores per strip) of B. subtilis var. niger are placedthroughout, in and/or associated with, the contaminated structure inorder to evaluate the effectiveness of the decontamination process aftercompletion. Similar, if not identical spore strips were used as testmarkers for monitoring the effectiveness of the chlorine dioxidefumigation that was used in an attempt to decontaminate parts of theHart Senate Building in December 2001. These strips are also standardindicators for verifying the efficacy of chemical, dry-heat, and steamsterilization equipment that is frequently used in dental and medicalfacilities. The 10⁸ Bacillus subtilis var. niger spore strips arederived from ATCC (American Type Culture Collection, the repositorywhere spore crops are obtained) #9372 and can be purchased from RavenBiological Laboratories in Omaha, Nebr. Upon completion of thesterilization or decontamination process, the strips are recovered andsent to a laboratory where they are incubated in a tryptic soy broth forculturing. After a certain period of time, typically 48 hours, a colorchange of the broth, for example from purple to yellow, indicates sporegermination and vegetative growth of the Bacillus. If however, after oneweek the broth color does not change, then the spores are not viable andthe decontamination and/or sterilization process is successful.

It is understood by those skilled in the art that the dosage of afumigant, and hence its effectiveness, is expressed by the product ofthe fumigant concentration and the time of exposure, commonly calledconcentration and time product, abbreviated as CT. The unit of CT ismg-hr/l if expressed in metric system. However, it is found that for thepurpose of the present invention CT is not the dominant factor forachieving decontamination of pathogenic microorganisms, particularlybacteria spores. Instead, concentration of the methyl bromide gas andtemperature used for the exposure play more critical roles.

Examples 1, 2 and 3 illustrate the functions of temperature and theconcentration of methyl bromide under laboratory test conditions.Example 4 illustrates the functions of temperature and the concentrationof methyl bromide under field test conditions. As shown in Table 1, attemperature of 19° C. and methyl bromide concentrations from 48 to 80mg/l (theoretical value), B. subtilis spores, after an exposure time aslong as 164 hours, still germinated. Therefore, the decontamination wasnot successful. On the contrary, as shown in Table 2, when the exposuretemperature was raised to 27° C., with a methyl bromide concentration of320 mg/l (theoretical value) B. subtilis spore germination wassuccessfully disabled, after exposure under such a condition for 72hours. Moreover, as shown in Table 3, when the exposure temperature wasfurther raised to 32° C., with a methyl bromide concentration of 240mg/l (theoretical value) B. subtilis spore germination was successfullydisabled, after exposure under such a condition for only 38 hours.However, at the same temperature (32° C.) a methyl bromide concentrationof 160 mg/l (theoretical value) was not sufficient to stop germinationof the spores. In addition, as shown in Table 4, when in a fieldenvironment the exposure temperature was put in the range of 28° C. to38° C., with a methyl bromide concentration of 250 to 350 oz/mcf,germinations of B. subtilis and B. stearo. at concentrations of 10⁶ and10⁵, respectively were successfully disabled, after exposure under theseconditions for 48 hours. Moreover, germinations of these spores instrips with concentrations of 10⁸, were also successfully disabled afterexposure under these conditions for 48 hours.

Furthermore, Example 2 also illustrates the effect of methyl bromideexposure to office commodities commonly present in office buildings. Theresults showed that with 96 hours exposure at 27° C. to a methyl bromideconcentration as high as 320 mg/l, no observable damage had occurred tothe office commodities. Unaltered data were recovered from the storagemedia (video tape, CD, and Zip diskette), the calculator functioned, andthe unexposed film was exposed and developed without damage. Thisdemonstrated utility of the method of the present invention fordecontaminating whole structures, such as office buildings and commonoffice commodities contained therein.

It is noted methyl bromide concentrations listed in Tables 1-3 wereobtained from the theoretical calculation based on syringe-injectedvolume of the methyl bromide gas. As stated in the examples, uponincreasing the temperature and re-equilibrating the chambers toatmospheric pressure, there is an estimated loss of methyl bromide ofabout 8%. Furthermore, it is also known that up to 6% of methyl bromidecan be absorbed by the structure under the decontamination treatment.Therefore, the airborne concentration of methyl bromide retained in thesealed structure is about 8 to 15% lower than the theoretical value.More specifically, for a theoretical value of 240 mg/l, the actualconcentration is between 204 mg/l and 220 mg/l.

Therefore, for the purpose of the present invention it is preferred tocarry out the decontamination process at a temperature greater than 20°C., more preferably at a temperature greater than 25° C., and mostpreferably at a temperature about 27° C. and above. On the other hand,the concentration of methyl bromide used for the decontamination ofpathogenic microorganisms is preferably about 200 mg/l and above. Inaccordance with the data obtained from the field test described inExample 4, additional optimal constraints for temperature, time andconcentration of the fumigation are also described below.

In the fumigation industry, the amount of methyl bromide required toachieve a certain target concentration is calculated first. Afterintroducing the calculated amount of methyl bromide into a sealedstructure, the actual methyl bromide concentration is measured using gasdetectors described hereinafter. During the fumigation, the methylbromide concentration can be monitored frequently, and can be maintainedat the desired level by refill if it is needed.

Since the effective exposure temperatures in the range of greater than20° C. to about 38° C. can be conveniently achieved in the field, andsince introducing methyl bromide gas into a sealed structure has beenpracticed many years in the fumigation industry, the effectivedecontamination method demonstrated in the laboratory scale can beimplemented conveniently in the field by using the structural fumigationtechniques. It is known to those in the fumigation industry, thesetechniques require that the building exterior be completely covered withtarpaulins that are clamped together at their seams and sealed at groundlevel with “sand snakes”. Some buildings with gas tight outer walls androof structures can be sealed using polyethylene sheeting taped overventilation systems, windows, and other locations where gas mightdiffuse from the building. Once the building is sealed, methyl bromideis added by allowing the pressurized liquid in methyl bromide cylindersto pass through a heat exchanger where the methyl bromide becomesgaseous and from there it is introduced by hose into the buildinginterior. Methyl bromide concentration can be then monitored using athermal conductivity detector (Fumiscope®, from Key Chemical &Equipment, Clearwater, Fla.) throughout the fumigation operation. Ifconcentrations are found to fall below target amounts during thefumigation, additional methyl bromide is added. After the fumigation,the seal is removed and the structure aerated. It is apparent that thesame techniques can also be used for decontaminations of vehicles,trains, boats, or airplanes.

It is known in the fumigation industry that once airborne methyl bromideconcentrations are below 3 ppm, the treated building and its contentsare safe for reoccupation and for all normal activities. Post fumigationmethyl bromide concentration can be measured using gas detector tubes,such as Kitagawa gas detector tubes manufactured by Matheson SafetyProducts, East Rutherford, N.J., or a Miran® infrared gas-detectingdevice from Thermo Environmental Instruments, Franklin, Mass. Given thatmethyl bromide is already registered by the EPA as a structural andcommodity fumigant, it has long been determined that human contact withany of the particular contents of the building would pose no greaterrisk than contact with structural parts or surfaces of the buildingitself. Methyl bromide can be released effectively from the structure byaeration after the structure is unsealed. Further, currentinstrumentation already exists for field monitoring and clearing ofmethyl bromide without requirement for the use, as in the case of theprior art method of chlorine dioxide decontamination, of a one milliondollar piece of equipment known as a Trace Atmospheric Gas Analyzer.

Bacteria test strips used as a means to confirm the disabling ofgermination of the pathogenic microbes are placed in multiple locationsthroughout the structure inclusive of desks and office articles as wellas actual structural surfaces of the building itself. Therefore, thepresent inventive method differs from that of historical fumigation inits use in connection with microbes and the requirement for the use ofstrips of test bacteria spores to verify the effectiveness of any givenstructural cleanup.

In a further embodiment, the method of decontamination can be utilizedfor decontaminating articles contaminated by pathogenic microorganisms.A practical example of contaminated articles is packages of mailshandled daily in the post offices. The process can be easier for thearticles than for large structures because of their smaller sizes. Themethod comprises the steps of placing a contaminated article in a closedchamber, and sealing the chamber; introducing a methyl bromide gas intothe sealed chamber to a concentration of methyl bromide sufficient todeactivate the pathogenic microorganisms and disable germination of thepathogenic bacteria spores, and maintaining the concentration of methylbromide in the sealed chamber at a sufficient temperature for asufficient period of time, and resulting in deactivating the pathogenicmicroorganisms and disabling germination of the pathogenic bacteriaspores associated with the contaminated article. After thedecontamination, the chamber is unsealed, and the methyl bromide gas isreleased. The treated article can be retrieved from the chamber forreuse when the methyl bromide concentration is below 3 ppm.

As stated previously, B. subtilis spore test strips are a universallyaccepted standard for determination of whether a bacterial spore hasbeen deactivated by a treating agent or a treatment method. Therefore,based on the effectiveness demonstrated above, it is believed that themethod of the present invention has broad applications againstmicroorganisms, particularly Bacillus spores. More specifically, themethod of the present invention can be utilized to decontaminatestructures, or articles contaminated by pathogenic bacteria and theirspores including, but not limited to, Bacillus anthracis, and itsspores, B. subtilis var niger and its spores, and B. stearothermophilusand its spores.

In light of terrorism actions of September 2001, it is apparent that themethod of the present invention has a very important value in terms ofprotecting public safety by effectively controlling furthercontaminations, which can be potentially spread from contaminatedstructures, and/or articles. It also has important economic valuesbecause of recovering those contaminated structures, or importantarticles.

EXAMPLE 1

Spore Strips

The University of Florida Sterilization Monitoring and ConsultingService, Dept. of Oral & Maxillofacial Surgery, Gainesville, Fla.,provided combined species spores strips that contained 10⁶ spores of B.subtilis var. niger strips derived from ATCC #9372, and 10⁵ spores of B.stearothermophilus derived from ATCC #7953. The spore strips were inSchleicher & Schuell filter paper (#470, 6.4×38.1 mm) packaged in aGlassine® paper pouch. Glassine paper is permeable to sterilant butresistant to moisture and air at ambient temperature/pressure. Thesestrips are standard indicators for verifying the efficacy of chemical,dry-heat, and steam sterilization equipment. After the methyl bromideexposures, the strips were incubated in tryptic soy broth as recommendedby the strip manufacturer. At 48 hours, color change of the broth frompurple to yellow indicates spore germination and vegetative growth ofthe Bacillus. If broth color does not change, the spores are not viable.Germination results reported here are based on a one-week incubation attemperatures of 55° C. for B. stearothermophilus and 35° C. for B.subtilis var. niger. The incubations were performed by University ofFlorida for the 10⁶ spores of B. subtilis var. niger strips and 10⁵spores of B. stearothermophilus strips.

Methyl Bromide Exposures

Seven 9-liter glass desiccators were used as fumigation chambers (FIG.1). Each chamber was equipped with a septum port in the lid and a 12-cmpropeller attached to magnetic stir disk inside the base. Eachfumigation trial consisted of simultaneously exposing spore strips toseven methyl bromide exposure conditions. Two spore strips were placedin each chamber before chambers were sealed with silicone vacuum grease.The chamber was placed on a magnetic stirring plate that spun theinternal propeller at about 10 rps. Neat methyl bromide gas waswithdrawn from the headspace of a partially filled 150-ml Whitey(Highland Heights, Ohio) stainless steel sample cylinder filled from acommercial cylinder of Meth-O-Gas (>99.5% methyl bromide, Great LakesChemical Company, West Lafayette, Ind., Anon. 1989). The sample cylinderwas fitted with a Whitey DK series shut-off valve connected to a Kel-Fsyringe adapter with septum (Alltech Associates, Deerfield, Ill.).Headspace contaminants were purged by opening the valve and looseningand reseating the septum under a fume hood. Methyl bromide was thenwithdrawn through the septum with a gas syringe. The pressurized methylbromide gas in the syringe was allowed to reach equilibrium withatmospheric pressure by escaping from the needle after septum withdrawaland before the dense gas (3.874 mg/ml) was injected into chambers. Aftereach addition of 250 ml of methyl bromide gas, the chamber was relievedof pressure by inserting a syringe needle into the injection septum.After reaching temperature equilibrium, each chamber was again purged toequilibrate chamber pressure with atmospheric pressure. It was estimatedthat this pressure release resulted in a loss of no more than 8% of theintroduced methyl bromide. A previous study (Scheffrahn & Su,Comparative Toxicity Of Methyl Bromide Against Ten Nearctic TermiteSpecies (Isoptera: Termopsidae, Kalotermitidae, Rhinotermitidae). J.Econ. Entomol. 85:845-847, 1992) also showed methyl bromide loss bysorption of 6%. Chambers were held at 19° C. (room temperature) for 48to 164 hours. Relative humidity inside the chambers was about 40%.

The test results expressed as germination of Bacillus sp. spores afterexposure to methyl bromide in 9-liter glass chambers under selectedconcentration, temperature, and time conditions are shown in Table 1.

As shown at 19° C., Bacillus stearothermophilus spores were preventedfrom germinating at relatively low concentration of methyl bromide of 80mg/l. However, B. subtilis spores were much more resistant. At anincubation temperature of 19° C., with a concentration of methyl bromideas high as 320 mg/l (theoretical value) and exposure time as long as 164hours, B. subtilis spores were still able to germinate.

TABLE 1 MeBr Temp Conc. Time Dosage. Spore Germination (° C.¹) (mg/l²)(hours) (mg-hr/l) B. stearo. B. subtilis 19 48 63 3,000 yes yes 19 48104 5,000 yes yes 19 48 146 7,000 yes yes 19 80 112 9,000 no yes 19 80134 11,000  no yes 19 80 164 13,120  no yes 19 160 164 26,240  no yes¹Mean ± 0.4° C. ²Theoretical concentration based on methyl bromidevolume introduced. Actual concentration is lower.

EXAMPLE 2

The same materials and experiment conditions of Example 1 were used inthe Example 2, except the incubation temperatures and the concentrationsof methyl bromide in the chambers. The incubation temperatures wereraised up to 27° C. Methyl bromide concentration was raised up to 320mg/l (theoretical value). Furthermore, because B. sterothermophilusspores showed to be more susceptible to methyl bromide than those of B.subtilis under the experimental conditions of Example 1, both strips ineach chamber were incubated for B. subtilis in the experiment of Example2.

In addition to spore strips, one chamber (320 mg/l methyl bromide, 27°C., 96 hrs) was fitted with a videotape, CD with data, ZIP diskette withdata, calculator, AA battery, laser-printed document, disposable camerawith partially exposed film, galvanized and stainless steel washers, andphotographs (laser-jet and photo emulsion paper). Office commodityexposures were used to access the effect of methyl bromide on theseoffice commodities commonly present in office buildings.

The test results expressed as germination of Bacillus sp. spores afterexposure to methyl bromide in 9-liter glass chambers under selectedconcentration, temperature, and time conditions are shown in Table 2.

TABLE 2 MeBr Temp Conc. Time Dosage. Spore Germination (° C.¹) (mg/l²)(hours) (mg-hr/l) B. stearo. B. subtilis 20 240 48 11,520  nt³ yes 20320 72 23,040 nt yes 20 320 96 30,720 nt yes 27 240 48 11,520 nt yes 27320 72 23,040 nt no 27 320 96 30,720 nt no 27 320 96 30,720 nt  no⁴¹Mean ± 0.4° C. ²Theoretical concentration based on methyl bromidevolume introduced. Actual concentration is lower. ³nt = not tested. Inthese tests, both strips were tested for B. subtilis germination.⁴Chamber contained office commodities listed above.

As shown, when the exposure temperature was raised to 27° C. and with amethyl bromide concentration of 320 mg/l B. subtilis spore germinationwas successfully disabled, after exposure under such a condition for 72hours.

Furthermore, at 320 mg/l for 96 hours, no observable damage had occurredto the office commodities even though spore viability was inhibited.Unaltered data were recovered from the storage media (video tape, CD,and Zip diskette), the calculator functioned, and the unexposed film wasexposed and developed without damage.

EXAMPLE 3

The same materials and experiment conditions of Example 2 were used inthe Example 3, except that the incubation temperature was furtherelevated to 32° C.

The test results expressed as germination of Bacillus sp. spores afterexposure to methyl bromide in 9-liter glass chambers under selectedconcentration, temperature, and time conditions are shown in Table 3.

As shown, when the exposure temperature was further raised to 32° C.,with a methyl bromide concentration of 240 mg/l B. subtilis sporegermination was successfully disabled, after exposure under such acondition for only 38 hours, which was almost only half of the timeperiod required at 27° C. This experiment further illustrated theimportance of temperature in inhibiting germination of B. subtilisspores.

TABLE 3 MeBr Temp Conc. Time Dosage. Spore Germination (° C.¹) (mg/l²)(hours) (mg-hr/l) B. stearo. B. subtilis 27 320 48 15,360  nt³ no 27 32062 19,776 nt no 32 160 72 11,520 nt yes 32 240 48 11,520 nt no 32 240 7217,280 nt no 32 320 38 12,160 nt no 32 320 47 15,104 nt no ¹Mean ± 0.4°C. ²Theoretical concentration based on methyl bromide volume introduced.Actual concentration is lower. ³nt = not tested. In these tests, bothstrips were tested for B. subtilis germination.

It is noted, however, at 32° C. a methyl bromide concentration of 160mg/l with an exposure time of 72 hours is not sufficient to preventgermination of the B. subtilis spores. This suggests that the dosage(concentration and time product) is not predictive of efficacy. Exposuretemperature and methyl bromide concentration are more important inpreventing spore germination than exposure time.

While there has been shown and described the preferred embodiment of theinstant invention it is to be appreciated that the invention may beembodied otherwise than is herein specifically shown and described andthat, within said embodiment, certain changes may be made in the formand arrangement of the parts without departing from the underlying ideasor principles of this invention as set forth in the Claims appendedherewith.

EXAMPLE 4

Materials and Methods

Site Preparation. A partially furnished 10,000 ft³ mobile home situatedon a 100-acre State research facility was prepared for methyl bromide(MB) fumigation. The trailer was covered with 2 vinyl-coated nylontarpaulins that were clamped together and sealed at ground level withsand “snakes”. The trailer contained elements of both residential andoffice environments. The floor was carpeted, the walls were paneled withwood, and the ceiling was “popcorned”. Furnishings included a desk withdrawers, a bed, a plush reclining chair, cabinets, closets, and lightfixtures. Office furnishings included a Dell 133 MHz 586 personalcomputer (PC), electronic balance, videotape, CD with data, ZIP diskettewith data, hand calculator, “D” battery, laser-printed document,disposable camera with partially exposed film, 35 mm film slides,galvanized and stainless steel washers, copper-clad zinc pennies, chromeand steel clamp, photographs (color ink-jet and photo emulsion paper),newsprint, slick newsprint, magazines, and a black and white LaserJetdocument.

Monitoring lines were placed in the kitchen and bathroom to measure MBconcentration during the fumigation. The terminus of a “shooting” hosefrom which the MB was introduced was clamped inside a weighted bucket.One fan was placed in front of the “shooting” bucket and a second wasplaced in the hallway. Wireless temperature and humidity sensors(wireless weather station WS 2010, La Crosse, Technology Ltd., LaCrescent, Minn.) were placed at the kitchen ceiling, the kitchen floor,and the second bedroom closet. Temperature data loggers (Watch Dog 225and 450, Spectrum technologies, Inc., Plainfield, Ill.) were placed onthe arm of the reclining chair, the PC desk, the second bedroom windowair-conditioner, and the bathroom countertop. Oil-filled radiant heaters(Delonghi,1650 W) were placed in the living room, kitchen, firstbedroom, hallway, bathroom entry, and second bedroom.

Spore Strips. Combined-species spore strips (Raven BiologicalLaboratories, Omaha, Nebr.) with 10⁶ spores of B. subtilis var. nigerATCC # 9372 and 10⁵ of B. stearothermophilus ATCC #7953 were provided bythe University of Florida Sterilization Monitoring and ConsultingService, Dept. of Oral & Maxillofacial Surgery, Gainesville, Fla.Additionally, B. subtilis var. niger strips containing 10⁸ spores wereobtained directly from Raven Biological Laboratories. Spore strips werein Schleicher & Schuell filter paper (#470, 6.4×38.1 mm) packaged inGlassine® paper pouches. Glassine paper is permeable to a fumigant butresistant to moisture and penetration by external microbes. These stripsare standard indicators for verifying the efficacy of both chemical(ethylene oxide), dry-heat, and steam sterilization equipment. Thesestrips were also used to verify the sporicidal efficacy of chlorinedioxide in the 30,000 ft³ Daschle Suite of the Hart Senate Building.After the methyl bromide fumigation, the strips were incubated intryptic soy broth as recommended by the strip manufacturer. At 48 hours,color change of the broth from purple to yellow indicates sporegermination and vegetative growth of the Bacillus. If broth color doesnot change after 1 week, the spores are not viable and no germinationhas occurred. Germination results (pass=no growth, fail=growth) reportedherein (Table 4) are based on 1-week incubations at temperatures of 55°C. for B. stearothermophilus and 35° C. for B. subtilis.

Twenty sites within the trailer were selected that represented expectedand worst-case spore contamination sites (Table 4). At each site, twocombined-species strips and two 10⁸ B. subtilis strips were positionedeither by gravity or clear tape for a total placement of 80 sporestrips. A total of 40 additional control strips were stored at roomtemperature in the laboratory. After MB fumigation, all strips wererecovered from placement sites and returned, along with unfumigatedcontrol strips, to Gainesville and Omaha for incubation.

Fumigation and Aeration. Concentration, temperature, and time targets of320 oz/1,000 ft³, 95° F., and 48 hours, respectively, were selectedbased on conditions toxic to Bacillus subtilis spores. From 9:11-10:34on 21 Feb. 2002, 204.4 lbs of MB was introduced inside the trailer.Throughout the fumigation and aeration procedures, indoor concentrationof MB was measured from monitoring lines in the kitchen and bathroomusing a Fumiscope® thermal conductivity detector (Key Chemical andEquipment Co., Clearwater, Fla.) with an accuracy of 2 percent fullscale. At 48 hours post-equilibrium, a ca. 12 by 4 inch elliptical ventwas opened in a west-facing seam to begin the aeration process. MBmeasurements in the kitchen were taken hourly for the first 8 hoursduring which time the aeration vent was enlarged to 18 by 4 inches at 2hours into aeration, and ultimately, 36 by 10 inches at 6 hours intoaeration. At 24 hours into aeration, a fan was placed into the aerationvent and a second aeration vent was opened in the south seam. At 25hours after aeration was initiated, both tarpaulins were removed fromthe trailer to complete the residual aeration process.

Airborne MB Residues. During fumigation and aeration, MB residues weremonitored periodically at five locations: downwind at 20,100, and 150feet from the trailer, at College Avenue 140 ft due east of the trailer,and at the Florida Dept. of Forestry Fire facility 140 ft. southeast ofthe trailer. MB residues were measured using Kitagawa #157SB gasdetector tubes (0.4-80 ppm; Matheson Inc., East Rutherford, N.J.) and apump assembly. Measurements were taken 3 ft above ground and directlydownwind from the trailer or aeration vent while wearing aself-contained breathing apparatus.

Results and Discussion

Fumigation and Aeration. An equilibrium concentration of 335 oz/1,000ft³ (335 mg/l) was attained at 11:40 on February 21. As shown in FIG. 1,after the initial application of 204.4 lbs, three additionalapplications of 35.4, 35.4, and 31.3 lbs of MB were made on 21 February19:00, 22 February 7:15, and 22 February 18:00, respectively, tosupplement diffusive and sorptive losses for a total introduction of306.5 lbs. These cumulative applications resulted in corresponding peaksin MB concentration and yielded a time-weighted mean concentration of303.7 oz/1000 ft³ during the 48-hour fumigation for an accumulateddosage of 14,578 oz-hr/10,000 ft³ (mg-hr/l). To maintain a trailertemperature near 95° F., between 0-6 heaters were turned on as requiredto adjust for ambient temperature changes and radiant daytime heating asindicated by the wireless sensors. Temperatures reported in Table 4 wererecorded by the four temperature loggers placed in proximity of thespore strips. Mean relative humidity during the fumigation, recordedusing the bathroom logger with RH capability, was 62% (min. 50%, max.69%). During the fumigation, wireless sensors displayed the followingmax./min. temperature and % humidity values at three locations: kitchenceiling (T1, 100.4/90.5° F., 58/42%), kitchen floor (T2, 98.7/90.5° F.,59/42%), and second bedroom closet (T3, 100.7/92.1° F., 60/39%).

Aeration was initiated at 12:15 on February 23 when MB concentration inthe kitchen was 274 oz/1,000 ft³. Concentration steadily declined at arate of ca. 29 oz/1,000 ft³ per hour to 43 oz/1,000 ft³ at 20:15. At8:15 on 24 February, MB concentration was 4 oz/1,000 ft³ and at 24-hoursafter initiation of aeration, MB was not detected by the Fumiscope.

During the fumigation, a single detector tube reading of 2.5 ppm wasmeasured 20 ft downwind of the trailer (Table 5). All other pre-aerationreadings were between 0-1 ppm. As expected, aeration readings werehigher with the downwind readings at 20 feet measured at 5, 7.5, 45, 45,and 60 ppm. All readings were greatly influenced by erratic windmovements; however, readings at 100 ft and beyond did not exceed 3 ppmat any location.

Efficacy. Spore strips were recovered from the trailer sites after tarpremoval and MB aeration below 3 ppm on February 24 at 14:00. Four stripswere collected from each site and two of each type were placed inenvelopes that also contained a single control (unfumigated) stripstored in the laboratory. Fumigated and unfumigated strips wereincubation in Gainesville on February 25 and in Omaha on 26 February 02.In Gainesville, one strip from each site was incubated for B. subtilisgermination, the second strip for B. stearothermophilus.

Table 4 lists results of spore strip incubations. Spores on all of the10⁶ B. subtilis and all of the 10⁵ B. stearothermophilus strips werekilled by the MB fumigation indicating that conditions lethal to thebacterial spores were encountered at each of the 20 trailer sitestested. Each site also yielded at least one B. subtilis 10⁸ strip thatwas not viable after fumigation. Both B. subtilis 10⁸ strips produced novegetative cells during incubation at 16 sites. Only one 10⁸ strip eachsurvived in four other sites. Therefore, at 90% (16) of the sites, allfour test strips were killed, while at 10% of the sites 3 of 4 stripswere killed.

Collateral Damage. Equipment and materials were not damaged or otherwisevisually or functionally affected by exposure to MB during the trailerfumigation. The computer continued to run and process data normally, theundeveloped film was exposed and developed and prints appeared normal.The electronic balance functioned normally, and printed materialsremained as before the MB exposure. After aeration, however, a lingeringodor was detected inside the trailer for up to 7 days after thefumigation. The odor could be described as “tinny” ororganic—reminiscent of corn tortillas.

Comparison of MB with Chorine Dioxide. As of this writing, there are noUSEPA approved whole-structure remediation methods for Bacillus sporedecontamination of whole, large, furnished buildings. Although chlorinedioxide, in addition to other methods, was used to remediate the DaschleSuite, it is not practical for use as a whole-structure decontaminantbecause of collateral damage, inability to reach equilibrium, andinability to produce large quantities. MB, however, has been a provenand efficacious whole-structure fumigant for more than 50 years.Chlorine dioxide is also expensive, highly explosive, highly corrosive,and difficult to store.

Conclusions

This study demonstrates that Bacillus spores can be controlled with MBunder whole-structure conditions without any collateral damage. Minormodifications to existing MB fumigation methods include mean exposuretemperatures of at least 90° F. and concentrations approximating 300oz/1,000 ft³ for 48 hours.

TABLE 4 Spore strip location, proximal ambient temperature conditions,and incubation results (pass = no spore germination occurred; fail =spore germination occurred) for 80 strips at 303.7 oz/1,000 ft³ methylbromide exposure for 48 hours in trailer. Exposure Temp. ° F. 10⁶ 10⁵ B.subtilis 10⁸ Trailer Loc. & Descr. Mean Max. Min. B. sub. B. stear.Strip A Strip B 1-Floor vent, 95.12 98.8 89.6 pass pass pass fail insideducting 2-Under carpet fabric 95.12 98.8 89.6 pass pass pass pass3-Behind wall paneling 95.12 98.8 89.6 pass pass pass pass in insulation4-Wall plug outlet, 95.12 98.8 89.6 pass pass pass pass covered 5-Wallsurface, 95.12 98.8 89.6 pass pass pass pass in closed folder 6-Closedkitchen 95.07 98.8 89.6 pass pass pass pass cabinet 7-PC keyboard, 95.0798.8 89.6 pass pass pass pass inside back cover 8-PC CD tray, 95.07 98.889.6 pass pass pass pass closed 9-Desk drawer, 95.07 98.8 89.6 pass passpass pass closed 10-Desk drawer, 95.07 98.8 89.6 pass pass pass pass inclosed hanging file 11-Ceiling surface, 95.07 98.8 89.6 pass pass passpass exposed 12-Floor surface, 95.07 98.8 89.6 pass pass pass fail inclosed folder 13-Mattress, 95.07 98.8 89.6 pass pass fail pass under boxspring 14-Hall closet, 90.29 94.1 83.6 pass pass fail pass closed15-Medicine cabinet, 90.29 94.1 83.6 pass pass pass pass closed 16-Lightfixture, 90.29 94.1 83.6 pass pass pass pass secured globe 17-Central ACinlet, 90.29 94.1 83.6 pass pass pass pass behind filter 18-Window AC,93.24 100.2 88.2 pass pass pass pass behind filter 19-Under 93.24 100.288.2 pass pass pass pass newspapers 20-Recliner chair, 95.12 98.8 89.6pass pass pass pass under cover fabric

TABLE 5 Detector tube locations, wind conditions, and readings atvarious times during the trailer fumigation and aeration. Wind DistanceTube Wind Speed Direction from trailer reading Date Fumigation StatusTime Direction mph from trailer or vent ppm 21-Feb 4 hr into fumigation16:20 SSE 1-2 NNW 20 2.5 21-Feb 4 hr into fumigation 16:24 SSE 1-2 NNW100 0.7 21-Feb 4 hr into fumigation 16:30 SSE 1-2 NNW 150 trace 21-Feb 4hr into fumigation 16:35 — still E 140, street 0.0 21-Feb 4 hr intofumigation 16:39 — still SE 140, fire lab 0.0 22-Feb 18 hr intofumigation 6:38 — still N 20 1.0 22-Feb 18 hr into fumigation 6:45 —still N 100 0.0 22-Feb 18 hr into fumigation 6:54 — still E 140, street0.0 22-Feb 18 hr into fumigation 6:58 — still SE 140, fire lab 0.022-Feb 30 hr into fumigation 17:26 S 5-10 N 20 1.0 22-Feb 30 hr intofumigation 17:34 S 5-10 N 100 0.0 22-Feb 30 hr into fumigation 17:40 S5-10 E 140, street 0.0 22-Feb 30 hr into fumigation 17:44 S 5-10 SE 140,fire lab 0.0 23-Feb begin aeration 12:23 S 5-12 N 20 5.0 23-Feb beginaeration 12:35 S 5-12 N 100 1.2 23-Feb begin aeration 12:42 S 5-12 N 1501.2 23-Feb begin aeration 12:45 S 5-12 E 140, street 0.0 23-Feb beginaeration 12:50 S 5-12 SE 140, fire lab 0.0 23-Feb 1 hr into aeration13:18 SSW 10-20 NNE 20 7.5 23-Feb 1 hr into aeration 13:25 SSW 10-20 NNE100 3.0 23-Feb 1 hr into aeration 13:35 SSW 10-20 NNE 150 trace 23-Feb 1hr into aeration 13:40 SSW 5-15 E 140, street 0.0 23-Feb 1 hr intoaeration 13:45 SSW 5-15 SE 140, fire lab 0.0 23-Feb 2 hr into aeration14:18 var. SW 15-20 NE 20 45.0 23-Feb 2 hr into aeration 14:25 var. SW15-20 NE 100 1.5 23-Feb 2 hr into aeration 14:34 var. SW 15-20 NE 1501.0 23-Feb 2 hr into aeration 14:38 WSW 10-20 E 140, street 0.0 23-Feb 2hr into aeration 14:45 WSW 10-20 SE 140, fire lab 0.0 23-Feb 4 hr intoaeration 16:10 var. 0-3 N 20 45.0 23-Feb 4 hr into aeration 16:18 SSW0-3 N 100 1.0 23-Feb 4 hr into aeration 16:22 var. 0-5 NE 150 0.0 23-Feb4 hr into aeration 16:30 W 0-5 E 140, street 0.0 23-Feb 4 hr intoaeration 16:34 WSW 0-5 SE 140, fire lab 0.0 23-Feb 8 hr into aeration20:15 N 5-20 SE 20 60.0 23-Feb 8 hr into aeration 20:24 N 5-20 E 100 0.023-Feb 8 hr into aeration 20:28 N 5-20 SE 150 0.0 23-Feb 8 hr intoaeration 20:35 N 5-20 E 140, street 0.0 23-Feb 8 hr into aeration 20:40N 5-20 SE 140, fire lab 0.0

We claim:
 1. A method of decontaminating a structure contaminated bypathogenic microorganisms comprising the steps of: (a) sealing acontaminated structure sufficiently to enable retention of a gas, (b)introducing methyl bromide gas into sealed contaminated structure to aconcentration of methyl bromide in an amount sufficient to deactivatesaid pathogenic microorganisms and disable germination of pathogenicbacteria spores, and (c) maintaining said sealed contaminated structurewith said concentration of methyl bromide at a sufficient temperaturefor a sufficient period of time, and resulting in deactivating saidpathogenic microorganisms and disabling germination of said pathogenicbacteria spores associated with said contaminated structure.
 2. Themethod of decontaminating a structure of claim 1 further comprising thestep of unsealing and aerating decontaminated structure to release saidmethyl bromide gas for reuse of said structure.
 3. The method ofdecontaminating a structure of claim 1, wherein said pathogenic bacteriaand spores of said pathogenic bacteria comprise bacillus anthracis andits spores, B. subtilis var niger and its spores, and B.stearothermophilus and its spores.
 4. The method of decontaminating astructure of claim 1, wherein said structure comprises a room, aresidential or commercial building, a mobile home, a vehicle, a train, aboat, and an airplane.
 5. The method of decontaminating a structure ofclaim 1, wherein said sufficient temperature is greater than 20° C. 6.The method of decontaminating a structure of claim 5, wherein saidsufficient temperature is greater than 25° C.
 7. The method ofdecontaminating a structure of claim 5, wherein said sufficienttemperature is approximately between 25° C. and 40° C.
 8. The method ofdecontaminating a structure of claim 6, wherein said concentration ofmethyl bromide is about 200 mg/l and above.
 9. The method ofdecontaminating a structure of claim 1, wherein said concentration ofmethyl bromide is about 250 mg/l and above.
 10. The method ofdecontaminating a structure of claim 1, wherein said concentration is amean concentration of approximately 303 mg/l during the decontamination.11. The method of decontaminating a structure of claim 8, wherein saidsufficient time is greater than 30 hours.
 12. The method ofdecontaminating a structure of claim 9, wherein said sufficient time isapproximately 48 hours.
 13. The method of decontaminating a structure ofclaim 1 further comprising introducing an additive into said sealedcontaminated structure.
 14. The method of decontaminating a structure ofclaim 13, wherein said additive is chloropicrin.
 15. A method ofdecontaminating an article contaminated by pathogenic microorganismscomprising the steps of: (a) placing said contaminated article in aclosed chamber, and sealing said chamber, (b) introducing methyl bromidegas into sealed chamber to a concentration of methyl bromide sufficientto deactivate said pathogenic microorganisms and disabling germinationof said pathogenic bacteria spores, and (c) maintaining saidconcentration of methyl bromide in said sealed chamber at a sufficienttemperature for a sufficient period of time, and resulting indeactivating said pathogenic microorganisms and disabling germination ofsaid pathogenic bacteria spores associated with said contaminatedarticle.
 16. The method of decontaminating an article of claim 15further comprising the step of releasing said methyl bromide gas fromsaid chamber for reuse of said article.
 17. The method ofdecontaminating an article of claim 15, wherein said pathogenicmicroorganisms and pathogenic bacteria spores comprise bacillusanthracis and its spores, B. subtilis var niger and its spores, and B.stearothermophilus and its spores.
 18. The method of decontaminating anarticle of claim 15, wherein said sufficient temperature is greater than20° C.
 19. The method of decontaminating an article of claim 18, whereinsaid sufficient temperature is greater than 25° C.
 20. The method ofdecontaminating an article of claim 15, wherein said sufficienttemperature is approximately between 25° C. and 40° C.
 21. The method ofdecontaminating an article of claim 19, wherein said concentration ofmethyl bromide is about 200 mg/l and above.
 22. The method ofdecontaminating an article of claim 21, wherein said concentration ofmethyl bromide is about 250 mg/l and above.
 23. The method ofdecontaminating an article of claim 15, wherein said concentration is amean concentration of approximately 303 mg/l during the decontamination.24. The method of decontaminating an article of claim 21, wherein saidsufficient time is greater than 30 hours.
 25. The method ofdecontaminating an article of claim 23, wherein said sufficient time isapproximately 48 hours.
 26. A method of decontaminating a structurecontaminated by pathogenic microorganisms comprising the steps of: (a)sealing a contaminated structure sufficiently to enable retention of agas, (b) introducing methyl bromide gas into sealed contaminatedstructure to achieve a concentration of methyl bromide about 200 mg/land above, and (c) maintaining said sealed contaminated structure withsaid concentration of methyl bromide at a temperature greater than 25°C. for a sufficient period of time, and resulting in deactivating saidpathogenic microorganisms and disabling germination of said pathogenicbacteria spores associated with said contaminated structure.
 27. Themethod of decontaminating a structure of claim 26, wherein saidsufficient time is greater than 30 hours.